Rabbit polyclonal antibodies against mSYD1A were raised against a synthetic peptide (MAEPLLRKTFSRLRGREK) and CH5424802 molecular weight affinity purified on the antigen. Anti-pan-neuroligin was described previously (Taniguchi

et al., 2007). Rabbit anti-munc18 was a gift from Matthijs Verhage (de Vries et al., 2000). Other antibodies were purchased from commercial sources: mouse anti-actin (clone AC-40, Sigma-Aldrich), goat anti-cyclinA (#sc-31086, Santa-Cruz), mouse anti-PSD95 (#73-028, Neuromab), mouse anti-VAMP2 (clone 69.1, Synaptic Systems), anti-vesicular glutamate transporter 1 (vGluT1, #1353303, Synaptic Systems), rabbit anti-GAPDH (#E1C604, Enogene), mouse anti-CASK (#75-000, Neuromab), rabbit anti-munc13-1 (#126103, Synaptic Systems), rabbit anti-munc18-1 (#116002, Synaptic Systems), mouse anti-beta-tubulin (E7, DSHB), rabbit anti-ELKS 1b/2 (#143003, Synaptic Systems), rat anti-HA (clone 3F10, Roche Applied Science), rabbit anti-c-myc (#sc-789, Santa-Cruz), mouse anti-flag (#F1804, Sigma), rabbit anti-homer (#160003, Synaptic Systems), mouse anti-bassoon (#GTX13249, GeneTex), rabbit anti-calbindin (#CB38a, Swant), mouse anti-NeuN (#MAB377, Chemicon). Secondary

antibodies conjugated to cyanine dyes or Alexa 488 or 643 (Jackson ImmunoResearch and Invitrogen) were used for visualization in immunostainings. Candidate intrinsically disordered protein domains were predicted using the PrDOS server, an online tool that combines local amino acid information and template protein references (http://prdos.hgc.jp/cgi-bin/top.cgi) (Ishida and Kinoshita, 2008). The thermostability test for confirmation of intrinsically OSI-744 mw disordered sequences was performed as described previously (Galea et al., 2006). Briefly, HEK293T cells on 10 cm diameter dishes were transfected with expression constructs for mSYD1A. After 24 hr, cells were harvested in PBS with a cell scraper and resuspended

in 300 μl of Buffer A (10 mM sodium phosphate buffer [pH 7.0], 50 mM NaCl, 50 mM DTT, 0.1 mM sodium orthovanadate, complete protease inhibitor Roche]). Cells were mechanically cracked by passing L-NAME HCl through a 25G needle and centrifuged at 16,000 × g for 30 min at 4°C. The supernatant was transferred to a fresh tube and the protein concentration was adjusted to 1 mg/ml with Buffer A. The cell lysate was heated for 30 min or 1 hr at 90°C. The protein mixture was placed on ice for 15 min and centrifuged at 16,000 × g for 30 min at RT. Soluble proteins in the supernatant were precipitated with 10% trichloracetic acid. The pellets were resuspended in SDS-PAGE buffer and analyzed by western blotting. Förster-resonance energy transfer (FRET) assays were performed as described previously (Itoh et al., 2002). Briefly, HEK293T cells were transfected with the RhoA sensor (Pertz et al., 2006) and expression constructs of interest. After 48 hr cells were suspended in 1× PBS.